Simcha I, Shtutman M, Salomon D, Zhurinsky J, Sadot E, Geiger B, Ben-Ze'ev A
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Cell Biol. 1998 Jun 15;141(6):1433-48. doi: 10.1083/jcb.141.6.1433.
beta-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors. Here we compared the nuclear translocation and transactivation abilities of beta-catenin and plakoglobin in mammalian cells. Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates. The beta-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker. Moreover, transfection of LEF-1 translocated endogenous beta-catenin, but not plakoglobin to the nucleus. Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or beta-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1- responsive transcription was significantly higher with beta-catenin. Overexpression of wild-type plakoglobin or mutant beta-catenin lacking the transactivation domain induced accumulation of the endogenous beta-catenin in the nucleus and LEF-1-responsive transactivation. It is further shown that the constitutive beta-catenin-dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or alpha-catenin. The results indicate that (a) plakoglobin and beta-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1-dependent transactivation is preferentially driven by beta-catenin; and (c) the cytoplasmic partners of beta-catenin, cadherin and alpha-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity.
β-连环蛋白和桥粒斑珠蛋白是同源蛋白，它们通过将钙黏着蛋白与细胞骨架相连来发挥细胞黏附功能，并与淋巴增强结合/T细胞（LEF/TCF）转录因子一起通过反式激活参与信号传导。在此，我们比较了β-连环蛋白和桥粒斑珠蛋白在哺乳动物细胞中的核转位及反式激活能力。在MDCK细胞中过表达这两种蛋白中的任何一种都会导致核转位并形成核聚集体。含有β-连环蛋白的核结构中还含有LEF-1和纽蛋白，而桥粒斑珠蛋白在募集这些分子方面效率较低，这表明其与LEF-1和纽蛋白的相互作用明显较弱。此外，转染LEF-1可使内源性β-连环蛋白转位至细胞核，但不能使桥粒斑珠蛋白转位。由Gal4 DNA结合结构域和桥粒斑珠蛋白或β-连环蛋白的反式激活结构域组成的嵌合体在反式激活Gal4反应性报告基因方面同样有效，而β-连环蛋白对LEF-1反应性转录的激活明显更高。过表达野生型桥粒斑珠蛋白或缺乏反式激活结构域的突变型β-连环蛋白会诱导内源性β-连环蛋白在细胞核中积累以及LEF-1反应性反式激活。进一步研究表明，在SW480结肠癌细胞中，组成型β-连环蛋白依赖性反式激活及其核定位可通过过表达N-钙黏着蛋白或α-连环蛋白来抑制。结果表明：（a）桥粒斑珠蛋白和β-连环蛋白在核转位以及与LEF-1和纽蛋白形成复合物方面存在差异；（b）LEF-1依赖性反式激活优先由β-连环蛋白驱动；（c）β-连环蛋白的细胞质伙伴钙黏着蛋白和α-连环蛋白可将其隔离在细胞质中并抑制其转录活性。
